Biogeosciences, 11, 3531–3545, 2014
www.biogeosciences.net/11/3531/2014/
doi:10.5194/bg-11-3531-2014
© Author(s) 2014. CC Attribution 3.0 License.
High temperature decreases the PIC / POC ratio and increases
phosphorus requirements in Coccolithus pelagicus (Haptophyta)
A. C. Gerecht1 , L. Šupraha2 , B. Edvardsen3 , I. Probert4 , and J. Henderiks1,2
1 CEES,
Dept. of Biosciences, University of Oslo, P.O. Box 1066 Blindern, 0316 Oslo, Norway
of Earth Sciences, Palaeobiology, Villavägen 16, 75236 Uppsala, Sweden
3 Marine Biology, Dept. of Biosciences, University of Oslo, P.O. Box 1066 Blindern, 0316 Oslo, Norway
4 UPMC, CNRS, Biological Station Roscoff, Place Georges Teissier, 29680 Roscoff, France
2 Dept.
Correspondence to: A. C. Gerecht (a.c.gerecht@ibv.uio.no)
Received: 22 November 2013 – Published in Biogeosciences Discuss.: 16 January 2014
Revised: 11 April 2014 – Accepted: 15 May 2014 – Published: 3 July 2014
Abstract. Rising ocean temperatures will likely increase
stratification of the water column and reduce nutrient input
into the photic zone. This will increase the likelihood of nutrient limitation in marine microalgae, leading to changes
in the abundance and composition of phytoplankton communities, which in turn will affect global biogeochemical
cycles. Calcifying algae, such as coccolithophores, influence the carbon cycle by fixing CO2 into particulate organic
carbon through photosynthesis (POC production) and into
particulate inorganic carbon through calcification (PIC production). As calcification produces a net release of CO2 ,
the ratio of PIC to POC production determines whether
coccolithophores act as a source (high PIC / POC) or a sink
(low PIC / POC) of atmospheric CO2 . We studied the effect
of phosphorus (P-) limitation and high temperature on the
physiology and the PIC / POC ratio of two subspecies of Coccolithus pelagicus. This large and heavily calcified species is
a major contributor to calcite export from the photic zone into
deep-sea reservoirs. Phosphorus limitation did not influence
exponential growth rates in either subspecies, but P-limited
cells had significantly lower cellular P-content. One of the
subspecies was subjected to a 5 ◦ C temperature increase from
10 ◦ C to 15 ◦ C, which did not affect exponential growth
rates either, but nearly doubled cellular P-content under both
high and low phosphate availability. This temperature increase reduced the PIC / POC ratio by 40–60 %, whereas the
PIC / POC ratio did not differ between P-limited and nutrientreplete cultures when the subspecies were grown near their
respective isolation temperature. Both P-limitation and elevated temperature significantly increased coccolith malfor-
mations. Our results suggest that a temperature increase may
intensify P-limitation due to a higher P-requirement to maintain growth and POC production rates, possibly reducing
abundances in a warmer ocean. Under such a scenario C.
pelagicus may decrease its calcification rate relative to photosynthesis, thus favouring CO2 sequestration over release. It
seems unlikely that P-limitation by itself causes changes in
the PIC / POC ratio in this species.
1
Introduction
Coccolithophores represent a prominent functional group of
marine phytoplankton and are major contributors to the carbon cycle. These eukaryotic microalgae fix CO2 into particulate organic carbon through photosynthesis (POC production) and into particulate inorganic carbon through calcification (PIC production). Although removing carbon from
seawater, the production of calcite scales (coccoliths) is a
net source of CO2 to the environment (Gattuso et al., 1995;
Rost and Riebesell, 2004). Therefore, the ratio of calcification to photosynthesis (PIC / POC production ratio) determines whether coccolithophores act as a source or a sink of
atmospheric CO2 (Balch et al., 1991; Holligan et al., 1993;
Buitenhuis et al., 1996). The ballasting of organic matter
(POC) by coccoliths (PIC) is believed to be an efficient way
of transporting carbon out of the photic zone (Armstrong
et al., 2002; Klaas and Archer, 2002), contributing to the
drawdown of atmospheric CO2 on longer time scales. In
carbon cycle models, the PIC / POC ratio is therefore used
Published by Copernicus Publications on behalf of the European Geosciences Union.
3532
A. C. Gerecht et al.: High temperature decreases the PIC / POC ratio
as a measure for carbon export into sedimentary reservoirs
(Archer, 1991; Ridgwell et al., 2009). To accurately model
the effect of a changing climate on ocean–atmosphere CO2
exchange, it is important to constrain the PIC / POC production ratio of coccolithophores.
Various environmental factors affect POC and PIC production in coccolithophores. Elevated CO2 may increase photosynthesis (Beardall and Raven, 2004) whereas calcification is
generally reduced at higher CO2 concentrations (Riebesell et
al., 2000; Feng et al., 2008; Findlay et al., 2011; Krug et al.,
2011). Rising ocean temperatures may directly impact POC
and PIC production in coccolithophores (Paasche, 2002) and
increase the likelihood of nutrient limitation via a more stratified water column (Sarmiento et al., 2004). In some cases,
nutrient limitation has been shown to increase the ratio of
PIC to POC production in the cosmopolitan species Emiliania huxleyi (Paasche and Brubak, 1994; Paasche, 1998; Riegman et al., 2000). In these studies, POC production rates decreased due to decreasing nutrient availability whereas PIC
production rates remained unaffected, leading to an increase
in the ratio of PIC to POC. In the field, the increased ratio
of loose coccoliths to coccospheres has also been ascribed
to decreasing nutrient availability (Balch et al., 1991; Fernández et al., 1993; van der Wal et al., 1995) and mesocosm
studies have shown that calcification continues after POC fixation ceases due to nutrient exhaustion (van Bleijswijk et al.,
1994). On the other hand, several other studies on E. huxleyi have reported stable PIC / POC ratios due to a decrease
in both POC and PIC production rates with decreasing nutrient availability (e.g. Fritz, 1999; Borchard et al., 2011) and a
decrease in the PIC / POC ratio under nutrient limitation has
also been reported (Langer et al., 2013a). A recent study by
Langer et al. (2012) examined the effect of P- and nitrogen
(N-) limitation on the PIC / POC ratio of Calcidiscus leptoporus and showed this ratio to be insensitive to limitation by
these macronutrients. Given the species- and strain-specific
response to ocean acidification (Langer et al., 2006, 2009;
Krug et al., 2011), it is not surprising that the response to
macronutrient limitation is not uniform among strains and
species. The response may also depend on the experimental
method, i.e. batch vs. (semi-) continuous cultures (Langer et
al., 2013a). In addition, only a few species have been studied
to date. It is therefore necessary to examine more species and
lineages of coccolithophores to gain a more general view of
the possible responses of coccolithophores to changing nutrient availability.
Coccolithus pelagicus (Wallich) Schiller, 1930 is one of
the largest and most heavily calcified extant coccolithophores
with a PIC / POC ratio of generally > 1.5 (Langer et al.,
2006; Krug et al., 2011). The calcite weights of individual
coccoliths are at least two orders of magnitude higher than
the average weight of coccoliths produced by the smaller
E. huxleyi (Beaufort and Heussner, 1999; Young and Ziveri,
2000; Cubillos et al., 2012). Hence, this species is an important contributor to calcite export into the sediments (Broerse
Biogeosciences, 11, 3531–3545, 2014
et al., 2000). Coccolithus pelagicus is divided into at least
two morphotypes on the basis of heterococcolith size and
distribution (Geisen et al., 2002; Sáez et al., 2003). Although
genetic information suggests that they are separated at the
species level (Sáez et al., 2003), we here use the division of
the two morphotypes into subspecies, C. pelagicus subsp.
braarudii (Gaarder) Geisen et al., 2002 and C. pelagicus
subsp. pelagicus, according to Geisen et al. (2002) as the
genetic and morphological differentiations are slight (Jordan
et al., 2004). The subarctic morphotype C. pelagicus subsp.
pelagicus can form substantial blooms in the North Atlantic
and North Pacific regions (e.g. Winter et al., 1994; Ziveri
et al., 2004), while the temperate morphotype C. pelagicus
subsp. braarudii is common in coastal upwelling regions of
the Northeast and Southeast Atlantic (e.g. Cachão and Moita,
2000; Henderiks et al., 2012).
We investigated the combined effect of P-limitation and
elevated temperature on the physiology, elemental quotas,
and the PIC / POC ratio of this species. One strain of each
subspecies was grown in batch culture under P-limited and
nutrient-replete conditions to test for physiological differences between the two subspecies. Subspecies pelagicus was
grown in P-limited and nutrient-replete batch culture at two
temperatures to test the effect of a temperature increase. Calcification was evaluated by the PIC quota of cells, individual
coccolith volumes and the occurrence of coccolith malformations. Whereas altered carbonate chemistry has been shown
to increase the malformation of coccoliths (e.g. Langer et al.,
2006, 2011; Rickaby et al., 2010), Langer et al. (2012) concluded that nutrient limitation does not significantly affect
coccolith morphology. Particulate organic carbon production
was monitored by growth rate and the POC quota of the cells.
2
2.1
Methods
Experimental design
Two strains of C. pelagicus were obtained from the Roscoff
Culture Collection. Strain RCC1200 is a clone of C. pelagicus subsp. braarudii and was isolated from the South Atlantic offshore Namibia. Strain RCC3776 is a clone of
C. pelagicus subsp. pelagicus and was isolated from the
North Atlantic near Scotland. Clonal batch cultures were
grown in triplicate in sterile-filtered modified K/2 medium
(Keller et al., 1987; with the following modifications: omission of Tris and Si, addition of NiCl2 × 6H2 O (3.14 nM),
increase of EDTA concentrations (5.85 µM)) at two initial
phosphate concentrations. Vitamins were added according
to f/2 medium (Guillard, 1975). Aged natural sea water
from the Oslo Fjord was enriched with 160 µM nitrate and
phosphate concentrations of either 10 µM (high-P treatment)
or 1 µM (low-P treatment). Low-P cultures were expected
to become P-limited, whereas the high-P control treatment
ensured nutrient-replete exponential growth throughout the
www.biogeosciences.net/11/3531/2014/
A. C. Gerecht et al.: High temperature decreases the PIC / POC ratio
experiment. Each subspecies was cultured near its respective
isolation temperature, subsp. braarudii at 15 ◦ C and subsp.
pelagicus at 10 ◦ C. The latter was also subjected to high-P
and low-P treatments at elevated temperature (15 ◦ C).
Cells were acclimated to experimental light and temperature conditions for at least 10 generations before starting the
experiments. Cultures were kept in culture flasks (350 mL,
BD Biosciences, USA) in an environmental test chamber
(MLR-350, Panasonic, Japan) on a 12:12 h light : dark cycle at an irradiance of ca. 100 µmol photons m−2 s−1 . Culture flasks were agitated manually on a daily basis. Two
experiments of paired high-P and low-P treatments (main
and additional experiments; see Tables 1–3) were carried out
for subsp. braarudii (15 ◦ C) and subsp. pelagicus (10 ◦ C).
Each high-P and low-P treatment consisted of three replicate cultures. Additionally, one paired high-P and low-P experiment was performed in triplicate with subsp. pelagicus
at 15 ◦ C. Cell densities were determined daily on an electronic particle counter (CASY, Roche Diagnostics, Switzerland) 2 hours after the onset of the light phase. Exponential growth rates (µmax ) were calculated by linear regression of log-transformed cell densities in exponential phase
over time. Low-P cultures were sampled upon reaching stationary phase (on the day that cell densities had increased
not more than 5 % compared to the previous day). High-P
cultures were sampled at similar cell densities while still in
exponential phase to ensure similar carbonate chemistry between the treatments.
High-P and low-P culture media were sampled for initial chemistry (T0 , Table 1). Upon sampling the cultures,
the growth media were again sampled to analyse carbonate
chemistry parameters and residual phosphate concentrations
at the time of sampling (Tsample , Table 1). Culture samples
were collected for elemental analysis (particulate organic
phosphorus (POP), particulate nitrogen (PN), POC, and total particulate carbon (TPC)), for morphological analysis of
coccoliths by polarized light (POL), and for measuring coccosphere diameters, coccolith coverage and coccolith malformations by scanning electron microscopy (SEM).
2.2
2.2.1
Medium chemistry
Residual phosphate
Culture media were sterile filtered (0.2 µm) into plastic scintillation vials (Kartell, Germany) and stored at −20 ◦ C until analysis. Orthophosphate concentrations were determined
colorimetrically on a spectrophotometer (UV 2550, Shimadzu, Japan) as molybdate reactive phosphate following
Murphy and Riley (1962) with a precision of ±4 %. Residual
phosphate concentrations near or below the detection limit
(ca. 0.05 µM) confirmed that low-P cultures had taken up all
available phosphate from the medium by the time of sampling (Tsample , Table 1), whereas phosphate concentrations
of high-P cultures remained > 6.4 µM.
www.biogeosciences.net/11/3531/2014/
2.2.2
3533
Carbonate chemistry
Samples for total alkalinity (AT ) and pH were filtered
through GF/F filters (Whatman, GE Healthcare, UK), stored
airtight at 4 ◦ C and analysed within 24 h. Total alkalinity
was calculated from Gran plots (Gran, 1952) after duplicate potentiometric titration using an automatic titrator (TTA
80 Titration assembly, Radiometer, Denmark) or manual
titration with a precision of ±50 µmol kg−1 . The pH was
measured with a combined electrode (Red Rod, Radiometer) which was two-point calibrated to NBS scale (precision ±0.4 %). The carbonate system was calculated using the
program CO2sys (version 2.1 developed for MS Excel by
D. Pierrot from E. Lewis and D. W. R. Wallace) using the
dissociation constants for carbonic acid of Roy et al. (1993).
2.3
2.3.1
Elemental quotas and ratios
Particulate organic phosphorus
Samples for POP were filtered onto precombusted (500 ◦ C,
2 h) GF/C filters (Whatman) and stored at −20 ◦ C. Particulate organic phosphorus was converted to orthophosphate by oxidative hydrolysis with potassium persulfate under high pressure and temperature in an autoclave (3150EL,
Tuttnauer, Netherlands) according to Menzel and Corwin (1965). This was then quantified as molybdate reactive
phosphate as described in Sect. 2.2.1.
2.3.2
Particulate total and organic carbon and nitrogen
Samples for TPC, POC and PN were filtered onto precombusted GF/C filters, dried at 60 ◦ C overnight in a drying oven
and stored in a desiccator until analysis on an elemental analyser (Flash 1112, Thermo Finnigan, USA; detection limit
2 µg) with a precision of ±8 %. Particulate inorganic carbon
was removed from POC filters by pipetting 230 µL of 2 M
HCl onto the filters before analysis (Langer et al., 2009) and
PIC calculated as the difference between TPC and POC. Data
for PIC was available only for the main experiments of subsp.
braarudii (15 ◦ C) and subsp. pelagicus (10 ◦ C and 15 ◦ C). As
production rates cannot be accurately calculated in batch cultures going into stationary phase due to discontinuous growth
rates (Langer et al., 2012, 2013a), we compare elemental ratios of P-limited and nutrient-replete cultures using cellular
quotas, rather than production rates. These were compared
using a t-test and one-way analysis of variance (ANOVA) in
GraphPad Prism version 6 for Windows (GraphPad Software,
USA). For high-P cultures (sampled in exponential phase),
production rates were calculated from exponential growth
rates (µmax ) and the respective cellular quota.
Biogeosciences, 11, 3531–3545, 2014
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A. C. Gerecht et al.: High temperature decreases the PIC / POC ratio
Table 1. Initial (T0 ) and residual (Tsample ) medium chemistry: phosphate concentrations and carbonate chemistry parameters in paired
high-P and low-P media batch experiments with Coccolithus pelagicus subsp. braarudii (RCC1200) grown at 15 ◦ C and subsp. pelagicus
(RCC3776) grown at 10 and 15 ◦ C. Initial values (T0 , n = 2) were measured directly from the high-P and low-P medium. The residual
medium chemistry represents the average of triplicate batch cultures (Tsample , n = 3) with standard deviation (SD) in brackets.
C. pelagicus
subsp. braarudii (RCC1200)
high-P 15 ◦ C
low-P 15 ◦ C
subsp. pelagicus (RCC3776)
high-P 10 ◦ C
low-P 10 ◦ C
high-P 15 ◦ C
low-P 15 ◦ C
T0
Tsample
T0
Tsample
T0
Tsample
T0
Tsample
T0
Tsample
T0
Tsample
7.96
7.10
(0.32)
1400
(50)
7.81
(0.02)
488
(13)
18.3
(0.5)
1250
(50)
61
(6)
1350
(100)
1.5
(0.2)
1.05
0.00
(0.00)
1500
(50)
7.92
(0.01)
369
(2)
13.9
(0.1)
1300
(50)
81
(3)
1350
(0)
2.0
(0.1)
9.75
9.28
(0.33)
1650
(50)
7.84
(0.03)
415
(18)
18.2
(0.8)
1450
(50)
76
(9)
1550
(50)
1.8
(0.2)
1.16
0.08
(0.08)
1350
(50)
7.91
(0.06)
285
(34)
12.5
(1.5)
1150
(50)
69
(7)
1250
(50)
1.7
(0.2)
9.55
6.40
(0.91)
1400
(50)
7.81
(0.02)
477
(11)
17.9
(0.4)
1250
(50)
60
(5)
1300
(50)
1.4
(0.2)
1.05
0.03
(0.05)
1550
(50)
7.95
(0.03)
367
(27)
13.8
(1.0)
1350
(50)
92
(2)
1450
(50)
2.2
(0.1)
(a) Main experiments
PO3−
4 (µM)
(SD)
AT (µmol kg−1 )
(SD)
pH, NBS
(SD)
pCO2 (µatm)
(SD)
CO2 (µmol kg−1 )
(SD)
−1
HCO−
3 (µmol kg )
(SD)
−1
CO2−
3 (µmol kg )
(SD)
DIC (µmol kg−1 )
(SD)
c
(SD)
2000
8.12
293
11.0
1600
161
1750
3.9
2000
7.98
433
16.3
1700
123
1850
3.0
2050
8.15
219
9.6
1600
171
1800
4.1
2050
8.20
190
8.4
1550
189
1750
4.5
1950
8.18
240
9.0
1500
175
1700
4.2
2000
7.99
421
15.8
1700
126
1850
3.0
(b) Additional experiments
PO3−
4 (µM)
(SD)
AT (µmol kg−1 )
(SD)
pH, NBS
(SD)
pCO2 (µatm)
(SD)
CO2 (µmol kg−1 )
(SD)
−1
HCO−
3 (µmol kg )
(SD)
−1
CO2−
3 (µmol kg )
(SD)
DIC (µmol kg−1 )
(SD)
c
(SD)
10.2
2200
8.15
296
11.1
1750
188
1900
4.5
7.08
(0.85)
1300
(100)
7.90
(0.01)
347
(21)
13.0
(0.8)
1100
(100)
66
(5)
1200
(100)
1.6
(0.1)
1.14
2100
8.15
284
10.7
1650
180
1850
4.3
0.06
(0.06)
1350
(50)
8.06
(0.01)
227
(2)
8.5
(0.1)
1100
(50)
95
(4)
1200
(50)
2.3
(0.1)
2.4 Coccosphere and coccolith morphology
2.4.1 Coccolith dimensions
Samples for POL were filtered onto cellulose nitrate filters
(0.8 µm, Whatman) after dispersing the coccoliths with a
Triton–NaOCl treatment (Paasche et al., 1996). Filters were
dried at 60 ◦ C in a drying oven, mounted with Canada Balsam (Merck, USA) on microscope slides and viewed unBiogeosciences, 11, 3531–3545, 2014
8.90
2050
8.15
219
9.6
1600
171
1800
4.1
7.70
(0.19)
1400
(50)
7.23
(0.01)
1751
(81)
77.0
(3.6)
1350
(50)
15
(1)
1450
(50)
0.4
(0.0)
1.11
2000
7.91
418
18.4
1750
104
1850
2.5
0.08
(0.07)
1350
(50)
7.86
(0.01)
315
(14)
13.9
(0.6)
1150
(50)
62
(3)
1250
(50)
1.5
(0.1)
der crossed polarizers with a DM6000B Leica microscope,
which had a modified turret accommodating polarizing filters at different angles (Beaufort et al., 2014). Individual
coccoliths were randomly selected and captured at 1000×
magnification with a SPOT Flex colour digital camera (Diagnostic Instruments Inc., USA) and subsequently processed to
a compound 8 bit greyscale image (Beaufort et al., 2014). A
custom-made macro in ImageJ freeware measured coccolith
length through an ellipse approximation, as well as mean
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A. C. Gerecht et al.: High temperature decreases the PIC / POC ratio
3535
Table 2. Cellular quotas and molar ratios derived from paired high-P and low-P media batch experiments (n = 3) of Coccolithus pelagicus
subsp. braarudii (RCC1200) grown at 15 ◦ C, and subsp. pelagicus (RCC3776) grown at 10 and 15 ◦ C. Note that cell concentrations reflect
those at time of sampling, and that maximum growth rate (µmax ) was calculated during exponential growth phase. Low-P cultures were in
stationary phase at time of harvest. Reported are the averages of triplicate batch cultures with standard deviation (SD) in brackets.
C. pelagicus
subsp. braarudii (RCC1200)
high-P 15 ◦ C low-P 15 ◦ C
subsp. pelagicus (RCC3776)
high-P 10 ◦ C low-P 10 ◦ C
high-P 15 ◦ C
low-P 15 ◦ C
8450
(350)
0.32
(0.02)
10.3
(0.7)
217
(14)
116
(48)
54
(7)
6.7
(0.7)
0.54
(0.19)
9650
(1200)
0.34
(0.03)
4.4
(0.5)
212
(21)
189
(28)
120
(28)
16
(4)
0.89
(0.04)
(a) Main experiments
Cell concentrations (cells mL−1 )
(SD)
µmax (d−1 )
(SD)
POP (pg cell−1 )
(SD)
POC (pg cell−1 )
(SD)
PIC (pg cell−1 )
(SD)
POC/POP (mol mol−1 )
(SD)
PN/POP (mol mol−1 )
(SD)
PIC / POC (mol mol−1 )
(SD)
12750
(650)
0.42
(0.03)
5.9
(0.3)
155
(11)
208
(24)
66
(6)
7.5
(0.5)
1.34
(0.05)
11 800
(500)
0.37
(0.03)
2.8
(0.4)
169
(9)
200
(18)
163
(28)
18
(3)
1.18
(0.08)
10 000
(1250)
0.24
(0.03)
5.9
(0.8)
245
(33)
312
(34)
107
(7)
9.7
(0.4)
1.28
(0.19)
13 200
(400)
0.36
(0.02)
2.6
(0.5)
229
(19)
334
(52)
229
(25)
23
(3)
1.45
(0.15)
17 550
(1300)
0.49
(0.02)
4.7
(0.6)
207
(9)
n/a
15 000
(1350)
0.52
(0.04)
2.3
(0.5)
231
(35)
n/a
16 400
(400)
0.35
(0.03)
5.0
(0.4)
371
(6)
n/a
13 650
(300)
0.29
(0.02)
2.6
(0.3)
437
(29)
n/a
114
(8)
12
(1)
n/a
262
(58)
25
(5)
n/a
193
(2)
22
(2)
n/a
433
(37)
56
(7)
n/a
(b) Additional experiments
Cell concentrations (cells mL−1 )
(SD)
µmax (d−1 )
(SD)
POP (pg cell−1 )
(SD)
POC (pg cell−1 )
(SD)
PIC (pg cell−1 )
(SD)
POC/POP (mol mol−1 )
(SD)
PN/POP (mol mol−1 )
(SD)
PIC / POC (mol mol−1 )
(SD)
grey level (MGL) of the selected area measured in intensity
values of 0 (black) to 255 (white). Mean grey level was used
as a proxy for a change in coccolith thickness based on the
birefringence of calcite (Beaufort, 2005). Coccolith length
measured by this method considers the proximal shield only
as the larger distal shield is non-birefringent under crossed
polarizers (e.g. Cubillos et al., 2012).
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2.4.2
Coccolith malformations and coccosphere
dimensions
Samples for SEM were filtered onto polycarbonate filters
(0.8 µm, Cyclopore, Whatman), air-dried and sputter-coated
with gold-palladium. Imaging was performed with an S-4800
field emission scanning electron microscope (Hitachi, Japan)
at 2500× magnification. Coccolith morphology was classified into four categories: normal, incomplete (normal, but
incomplete distal shield elements), malformed (more than
one malformed distal shield element), and very malformed
(blocky structures or holes). Coccosphere diameter and the
Biogeosciences, 11, 3531–3545, 2014
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A. C. Gerecht et al.: High temperature decreases the PIC / POC ratio
Table 3. Coccosphere and coccolith size parameters as well as coccolith morphology from paired high-P and low-P media batch experiments
(n = 3) with Coccolithus pelagicus subsp. braarudii (RCC1200) grown at 15 ◦ C, and subsp. pelagicus (RCC3776) grown at 10 and 15 ◦ C.
Reported is sample size (n), average value of pooled triplicate batch cultures with standard deviation (SD) or 95 % confidence interval (CI)
in brackets.
C. pelagicus
subsp. braarudii (RCC1200)
high-P 15 ◦ C low-P 15 ◦ C
subsp. pelagicus (RCC3776)
high-P 10 ◦ C low-P 10 ◦ C
high-P 15 ◦ C
low-P 15 ◦ C
(a) Main experiments
n (number of coccospheres)
coccosphere diameter [µm]
(SD)
coccolith number
(SD)
245
19.9
(1.9)
15
(3)
135
19.7
(2.3)
15
(4)
coccosphere size parameters
165
201
18.0
20.4
(1.8)
(1.8)
15
17
(3)
(3)
142
16.6
(1.8)
12∗
(3)
91
18.3
(1.9)
12∗
(3)
n (number of coccoliths)
coccolith length [µm]
(SD)
MGL [intensity pixel−1 ]
(SD)
165
9.7
(1.2)
113
(14)
166
9.8
(1.4)
111
(16)
coccolith size parameters
329
324
8.8
9.0
(1.1)
(1.0)
113
123
(11)
(12)
164
8.8
(1.2)
114
(12)
167
9.0
(1.2)
117
(13)
n (number of coccoliths)
normal [%]
(CI)
incomplete [%]
(CI)
malformed [%]
(CI)
very malformed [%]
(CI)
1706
42
(2)
28
(2)
22
(2)
8
(1)
1215
29
(3)
25
(2)
28
(3)
18
(2)
coccolith morphology
978
1386
43
38
(3)
(3)
16
18
(2)
(2)
34
35
(3)
(3)
7
9
(2)
(2)
1373
28
(2)
8
(1)
39
(3)
25
(2)
1329
18
(2)
5
(1)
35
(3)
42
(3)
(b) Additional experiments
n (number of coccospheres)
coccosphere diameter [µm]
(SD)
coccolith number
(SD)
180
20.0
(2.1)
16
(3)
n (number of coccoliths)
coccolith length [µm]
(SD)
MGL [intensity pixel−1 ]
(SD)
300
9.8
(1.1)
123
(12)
n (number of coccoliths)
normal [%]
(CI)
incomplete [%]
(CI)
malformed [%]
(CI)
very malformed [%]
(CI)
1412
30
(2)
33
(3)
27
(2)
10
(2)
coccosphere size parameters
152
53
21.0
18.6
(2.8)
(2.1)
15
16
(5)
(4)
coccolith size parameters
306
166
9.9
9.1
(1.1)
(1.1)
126
120
(12)
(12)
coccolith morphology
1568
413
30
38
(2)
(5)
22
20
(2)
(4)
23
35
(2)
(5)
25
7
(2)
(3)
77
19.3
(2.3)
18
(4)
165
8.8
(1.1)
124
(13)
568
32
(4)
12
(3)
39
(4)
17
(3)
∗ Many collapsed spheres.
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A. C. Gerecht et al.: High temperature decreases the PIC / POC ratio
3537
number of coccoliths covering one sphere were estimated
from the same calibrated SEM images.
3
3.1
Results
Phosphorus-limited growth
Division rates during exponential growth were comparable
between high-P and low-P treatments for both subspecies
(Table 2). The temperate morphotype subsp. braarudii
(RCC1200) had higher maximum growth rates (0.44 ± 0.06
d−1 ) than the subarctic morphotype subsp. pelagicus
(RCC3776) (0.32 ± 0.05 d−1 ). In low-P medium subsp.
braarudii grew to final cell concentrations of 1.3 ± 0.2 ×104
cells mL−1 (Table 2). Subspecies pelagicus grew to similar final cell concentrations of 1.3 ± 0.1 × 104 cells mL−1
at 10 ◦ C. At 15 ◦ C, however, stationary phase was reached
at lower cell densities (1.0 ± 0.1 × 104 cells mL−1 ) even
though exponential growth rates were the same between the
two temperatures. All low-P cultures were P-limited at the
time of sampling as demonstrated by lower POP quotas and
higher ratios of POC and PN to POP, compared to high-P
treatments (Table 2; Fig. 1a). The two subspecies had the
same POP quota when comparing low-P and high-P cultures, respectively. Particulate organic phosphorus production, however, was lower in subsp. pelagicus than in subsp.
braarudii because of lower growth rates (Fig. 1b). Most striking is the high POP quota of subsp. pelagicus cultures grown
at high temperature (15 ◦ C). This almost doubled in both
low-P and high-P treatments. Accordingly, POP production
rates in high-P cultures of subsp. pelagicus were more than
twice as high at 15 ◦ C.
The initial carbonate chemistry of the medium (T0 , Table 1) was modified by culture growth in all treatments with a
drawdown of alkalinity (AT ) of 25–41 % in subsp. braarudii
and of 20–34 % in subsp. pelagicus (Tsample , Table 1). The
availability of dissolved inorganic carbon (DIC) was reduced
by up to 37 % and there was a shift towards lower pH values in all cultures from an initial pH = 8.10 ± 0.09 to a minimum pH = 7.81. The high-P treatment of the additional experiment of subsp. pelagicus grown at 10 ◦ C showed a strong
shift in the system towards pH = 7.23 (Table 1b). The saturation state for calcite (c ) calculated for this treatment was
below one whereas all other treatments were saturated in calcite (c > 1, Table 1). This low pH value likely represents
a technical error (no other samples were measured on that
same day) rather than an actual shift in the system because
the culture parameters of this high-P treatment were otherwise very similar to the high-P treatment of the main experiment and there was no evidence for coccolith dissolution.
3.2
Cellular carbon quotas and ratios
The cellular POC quota was similar between low-P and highP treatments for each experiment of the two subspecies (twww.biogeosciences.net/11/3531/2014/
Figure 1. (A) Particulate organic phosphorus (POP) quotas of Coccolithus pelagicus subsp. braarudii grown at 15 ◦ C and subsp.
pelagicus grown at 10 and 15 ◦ C, in high-P and low-P medium.
(B) POP production in high-P cultures. Mean ± min/max; values
for subsp. braarudii at 15 ◦ C and subsp. pelagicus at 10 ◦ C are averages of the main and additional experiments (n = 6); for subsp.
pelagicus at 10 ◦ C, n = 3.
test: p > 0.05; Table 2), except for the additional experiment
of subsp. pelagicus grown at 10 ◦ C in which the cells of lowP cultures contained significantly more POC than those of
high-P cultures (t-test: p = 0.02; Table 2b). High temperature (15 ◦ C) did not affect POC quotas in either P-treatment
of subsp. pelagicus when only the main experiments were
considered (t-test: p > 0.05; Table 2a; Fig. 2a). In the additional experiments, POC quotas were higher in both low-P
and high-P treatments of both subspecies compared to the
main experiments (one-way ANOVA: p < 0.001; Table 2b).
These differences were not due to changing bacterial abundance as bacteria accounted for < 5 % of total organic carbon
in all cultures (data not shown). Interestingly, subsp. pelagicus cells contained almost 40 % more POC than cells of
subsp. braarudii, although subsp. pelagicus was smaller in
terms of coccosphere size (see Sect. 3.3). Particulate inorganic carbon quotas were not affected either, by P-limitation
(t-test: p > 0.05) when the cultures were grown near their respective isolation temperature. However, there was a significant reduction in the PIC quota of subsp. pelagicus at high
Biogeosciences, 11, 3531–3545, 2014
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A. C. Gerecht et al.: High temperature decreases the PIC / POC ratio
Figure 3. Particulate organic (POC) and particulate inorganic carbon (PIC) production of Coccolithus pelagicus subsp. braarudii
grown at 15 ◦ C and subsp. pelagicus grown at 10 and 15 ◦ C in highP medium. Mean ± min/max. Values are from main experiments
only, n = 3.
Figure 2. (A) Particulate organic carbon (POC) quotas of
Coccolithus pelagicus subsp. braarudii grown at 15 ◦ C and subsp.
pelagicus grown at 10 and 15 ◦ C, in high-P and low-P medium.
(B) Particulate inorganic carbon (PIC) quotas. (C) PIC / POC ratios.
Mean ± min/max. Values are from main experiments only, n = 3.
temperature (15 ◦ C) in both P-treatments (t-test: p < 0.001;
Fig. 2b). In consequence, the PIC / POC ratio of subsp. pelagicus was below one at 15 ◦ C compared to PIC / POC ratios of
1.37 ± 0.18 at 10 ◦ C (Fig. 2c). The decrease was strongest
(t-test: p = 0.009) in exponentially growing high-P cultures.
These had a PIC / POC ratio of 0.54 ± 0.19 compared to
a significantly higher PIC / POC ratio of 0.89 ± 0.04 in Plimited cultures at this temperature (t-test: p = 0.003). The
reduced PIC / POC ratio of high-P cultures of subsp. pelagicus at high temperature (15 ◦ C) was mainly due to the decreased rate of PIC production, although POC production
rate increased by ca. 20 % (Fig. 3).
3.3
Coccosphere and coccolith dimensions
The average coccosphere diameter of subsp. pelagicus
was smaller (18.1 ± 1.9 µm) than that of subsp. braarudii
(19.9 ± 2.0 µm) (Table 3; Fig. 4a). Phosphorus limitation
led to an increase in coccosphere size in subsp. pelagiBiogeosciences, 11, 3531–3545, 2014
cus, but not subsp. braarudii. Temperature, on the other
hand, decreased coccosphere size in subsp. pelagicus. These
smaller spheres were composed of fewer coccoliths (12 ± 3
coccoliths sphere−1 ) compared to spheres of high-P cultures
grown at 10 ◦ C (15 ± 3 coccoliths sphere−1 ).
In both subspecies, the mean length of the proximal
shield (PSL) was unaffected by changes in P-availability (ttest: p > 0.05; Fig. 4b). Temperature did not change mean
PSL in subsp. pelagicus either (one-way ANOVA: p > 0.05;
Fig. 4b). Coccoliths produced by subsp. pelagicus were significantly smaller than those produced by subsp. braarudii
(t-test: p < 0.001). Yet, PSL showed a wide range of 6.4–
14 µm in subsp. braarudii and 5.8–12 µm in subsp. pelagicus. Coccoliths of low-P cultures of subsp. pelagicus in the
main experiment had a significantly higher mean grey level
(MGL), a proxy for coccolith thickness (t-test: p < 0.0001;
Table 3a). However, this increase in thickness in low-P cultures could not be confirmed in the additional experiment or
between P-treatments at high temperature (15 ◦ C). The difference in mean coccolith thickness between P-treatments of
subsp. braarudii was < 3 %, which is not considered biologically significant (Table 3). There was no effect of temperature
on coccolith thickness (t-test: p > 0.05).
3.4
Coccolith malformations
Scanning electron microscopy revealed a background percentage of malformed coccoliths in high-P cultures of subsp.
braarudii of 30 and 37 % in the main and additional experiment, respectively (Table 3; Fig. 5). This percentage increased to 46–48 % under P-limitation, mainly due to an
increased presence of coccoliths with blocky structures or
holes, classified as “very malformed” (Table 3; Fig. 5). In
subsp. pelagicus, the background percentage of malformed
coccoliths in high-P cultures grown at 10 ◦ C was 42 % in
both experiments with a lower percentage of incomplete
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A. C. Gerecht et al.: High temperature decreases the PIC / POC ratio
3539
Figure 4. (A) Number of coccoliths per coccosphere plotted against the diameter of that coccosphere produced in high-P and low-P medium
by Coccolithus pelagicus subsp. braarudii grown at 15 ◦ C and subsp. pelagicus grown at 10 and 15 ◦ C. (B) Mean grey level (MGL) plotted
against mean proximal shield length (PSL) of individual coccoliths. Mean ± SD. Values for subsp. braarudii at 15 ◦ C and subsp. pelagicus
at 10 ◦ C are combined for the main and additional experiments.
coccoliths than subsp. braarudii (Table 3; Fig. 5). Malformations increased to 56 % under P-limitation in the additional,
but not in the main experiment. When subsp. pelagicus was
grown at elevated temperature (15 ◦ C), malformations increased to 64 % in high-P cultures. The highest abundance of
malformed coccoliths (77 %) was observed in subsp. pelagicus cultures grown under P-limitation and high temperature
with 42 % of coccoliths classified as “very malformed” (Table 3; Fig. 5).
4
Discussion
Laboratory experiments are useful to systematically
test the short-term physiological (plastic) response of
coccolithophores to various environmental factors and could
constrain how the PIC / POC (production) ratio is likely to
change under future climate scenarios. The experiments
presented here provide the first available data set to test the
effect of P-limitation and elevated temperature on C. pelagicus. We ran batch cultures of this species into P-limitation
at moderate final cell densities (ca. 13 000 cells mL−1 )
to obtain enough sampling material at feasible culture
volumes. The production of both POC and PIC created a
drift in the carbonate system during culture growth. The
(up to 37 %) consumption of DIC by the time of sampling
depended mainly on the final cell densities reached in the
culture. These final cell densities varied more strongly
between the main and additional experiments of the same
treatment (e.g. low-P) than between treatments (high-P vs.
low-P). Yet, the observed trends, such as increased coccolith
malformations in low-P cultures, were similar between
the experiments, making carbonate chemistry an unlikely
candidate for explaining the observed differences between
treatments. Although bicarbonate, a known substrate for
calcification, was reduced down to ca. 1100 µmol kg−1 ,
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Rickaby et al. (2010) have shown that a reduction in
bicarbonate down to 1000 µmol kg−1 had no effect on either
culture growth (same growth rate as control) or calcification
(low percentage of malformed coccoliths) in C. pelagicus
subsp. braarudii. The changes in calcification observed in
the current study (reduced PIC quota and increased coccolith
malformations) at high temperature were therefore not due
to reduced substrate availability as the cultures were kept
above the threshold for bicarbonate limitation. Furthermore,
due to lower final cell densities, bicarbonate availability was
actually higher in 15 ◦ C than in 10 ◦ C degree cultures of
subsp. pelagicus. The reduction in PIC quota at 15 ◦ C was
therefore not related to bicarbonate availability.
4.1
Effect of phosphorus limitation and temperature on
culture growth and phosphorus quotas
Initial phosphate concentrations of the growth medium did
not influence cell division in exponential phase in either subspecies. The exponential growth rate of subsp. braarudii
was similar to those determined previously under similar
experimental conditions (Taylor et al., 2007; Buitenhuis et
al., 2008). Although other studies have observed higher exponential growth rates (up to 0.9 d−1 ) for the same strain
(Langer et al., 2006; Krug et al., 2011), these authors used a
higher irradiance and a longer light phase which probably increased cell division rates. The subarctic subspecies (subsp.
pelagicus) had a lower exponential growth rate than the temperate subsp. braarudii, even when these two subspecies
were grown at the same temperature (15 ◦ C). This may be
due to strain-specific differences as have been described for
different strains of E. huxleyi (Langer et al., 2009).
We found clear evidence for P-limitation with significantly lower POP quotas and higher C/P and N/P ratios in
low-P cultures. The doubled POP quota of subsp. pelagicus grown at 15 ◦ C instead of 10 ◦ C, points towards higher
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A. C. Gerecht et al.: High temperature decreases the PIC / POC ratio
Figure 5. Morphology of coccoliths observed by scanning electron microscopy, shown as the percentage of four categories: normal (black),
incomplete (grey), malformed (white) and very malformed (red). (A) Main experiments and (B) additional experiments of high-P and low-P
cultures of Coccolithus pelagicus subsp. braarudii at 15 ◦ C and subsp. pelagicus at 10 and 15 ◦ C. Triplicate batch cultures (1, 2, 3) are
presented separately for each experiment.
P-requirements at elevated temperature. Because of this,
the same phosphate supply (1 µM) in low-P cultures supported lower final cell densities at 15 ◦ C although exponential growth rates were unaffected. This increased requirement
for P under temperature stress may be linked to increased
energy demands and deserves further study as an increased
P-requirement at high temperature could exacerbate nutrient
limitation in a warmer ocean (Sarmiento et al., 2004). Most
of cellular P is to be found in RNA (Geider and La Roche,
2002). The higher POP content of subsp. pelagicus grown at
high temperature (15 ◦ C) could therefore also be related to
an increased RNA content caused by upregulated expression
of genes related to a temperature stress response. However,
the nitrogen content of the cells did not increase, offering no
evidence for an increase in enzyme production.
4.2
Effect of phosphorus limitation and temperature on
the PIC / POC ratio
Temperature had a stronger effect on the PIC / POC ratio
than P-availability. Whereas P-limitation did not affect the
PIC / POC ratio when the subspecies were grown near their
respective isolation temperature, elevated temperature decreased the PIC / POC ratio by more than half in high-P culBiogeosciences, 11, 3531–3545, 2014
tures of subsp. pelagicus. This decrease in the PIC / POC ratio was driven by the 40–60 % decrease in PIC quota whereas
POC quotas were similar between the two temperatures. In
high-P cultures, PIC production declined by ca. 60 %. This
sharp reduction in PIC quota and production in subsp. pelagicus at elevated temperature, points towards a lower production of coccoliths or a decreased calcite content of single coccoliths. Although the variation in calcite content of individual coccoliths was high within one strain, the mean coccolith
calcite content was unaffected by the tested environmental
parameters (see also Sect. 4.3) and can therefore not explain
the reduced PIC quota. Light microscope images showed that
high-P cultures of subsp. pelagicus at elevated temperature
(15 ◦ C) contained ca. 12 % naked cells whereas no naked
cells where observed at 10 ◦ C. The coccolith coverage estimated by SEM was 12 coccoliths sphere−1 at 15 ◦ C compared to 15 coccoliths sphere−1 at 10 ◦ C, indicating reduced
coccolith coverage in the high-temperature treatment. Many
of the spheres observed in the high-temperature treatment of
both high-P and low-P cultures were collapsed and/or incomplete, increasing the uncertainty of the estimate for coccolith
coverage. However, total coccolith counts from POL samples
confirmed that cultures at 15 ◦ C contained ca. 40 % fewer
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A. C. Gerecht et al.: High temperature decreases the PIC / POC ratio
coccoliths than cultures at 10 ◦ C. Reduced PIC production
at elevated temperature was therefore due to a reduction in
coccolith production with cells being covered by fewer coccoliths and an increased percentage of naked cells. This, together with an increased percentage of malformed coccoliths,
apparently made the coccospheres unstable.
High temperature (15 ◦ C) changed the response of subsp.
pelagicus to P-limitation. At 15 ◦ C the PIC / POC ratio, although still lower than at 10 ◦ C, was significantly higher under P-limitation. This was due to a higher PIC quota of low-P
compared to high-P cultures. This observation seems counterintuitive considering that at 10 ◦ C there was no effect of Plimitation on the PIC quota in subsp. pelagicus. We suggest
that this difference lies in high-P and low-P cultures being
harvested in different growth phases (exponential vs. stationary phase) and the stronger dependence of POC production
on P-availability. We explain this hypothesis in more detail
in the following.
Coccolithus pelagicus has so far not been described to produce multiple coccolith layers (this study; Gibbs et al., 2013),
whereas E. huxleyi can more than double its PIC / POC ratio
by forming multiple layers of coccoliths around the cell (Linschooten et al., 1991; Paasche, 1998). It is therefore likely
that there is an upper constraint to the PIC / POC ratio in
C. pelagicus dictated by cell geometry. Without producing
multiple layers, this species cannot increase PIC quota without increasing the cell surface area necessary to accommodate further coccoliths. However, increasing the surface area
entails an increase in cell volume and thereby POC quota.
Under such a scenario, there is a limit as to how freely the
PIC / POC ratio can vary. This may explain the lack of increase in the PIC / POC ratio under P-limitation at “normal”
temperature (15 ◦ C for subsp. braarudii, 10 ◦ C for subsp.
pelagicus). Similarly, Langer et al. (2012) observed that the
PIC / POC ratio of C. leptoporus is stable under nutrient limitation. Riegman et al. (2000), on the other hand, reported
sustained PIC production in E. huxleyi during decreasing nutrient availability in P-limited chemostats in which POC production decreased.
The reason for the reduction in coccolith coverage in
subsp. pelagicus at elevated temperature (15 ◦ C) is unclear.
However, at this temperature the coccolith coverage of the
cells was overall reduced, alleviating the spatial constraint
to the production of new coccoliths. High-P cultures were
harvested in exponential phase, in which cells were dividing and POC production was relatively constant. Low-P cultures, on the other hand, were sampled in stationary phase
when cell division and POC production had declined due to
the depletion of phosphate from the medium. If we assume
PIC production to be less dependent on P-availability than
POC production, as suggested by Riegman et al. (2000) for
one particular strain of E. huxleyi, low-P cultures could have
continued producing coccoliths while cell division was slowing down. Sustained PIC production while POC production
was decreasing in cultures going into stationary phase would
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3541
explain the observed increase in the PIC / POC ratio in low-P
cultures.
4.3
Effect of phosphorus limitation and temperature on
coccolith morphology
The two subspecies overlapped in their size distribution,
both in regard to coccosphere diameter and coccolith length.
Overlapping coccolith size ranges have been previously described for these two subspecies, and our results confirm that
subsp. pelagicus is the smaller morphotype of the two (Baumann et al., 2000; Geisen et al., 2002; Cubillos et al., 2012).
There was no effect of either temperature or P-limitation
on the mean coccolith volume and the same amount of calcite was fixed into individual coccoliths independently of the
tested abiotic factors. Gibbs et al. (2013) have shown that
mean coccolith size in C. pelagicus subsp. braarudii does
not change during different growth phases and in E. huxleyi, coccolith dimensions have also been described as remaining constant among different dilution rates in N-limited
cyclostats (Fritz, 1999). It therefore seems that within one
strain, mean coccolith volume is not a plastic trait.
Using a conversion factor for mean grey level (MGL)
to coccolith thickness (Beaufort et al., 2014), we estimated mean coccolith calcite weight for the two subspecies.
Subspecies braarudii produced coccoliths with an average
weight of 117 ± 34 pg calcite whereas subsp. pelagicus coccoliths were lighter at 100 ± 27 pg calcite. These estimates
are lower than the estimates obtained using the shape factor of Young and Ziveri (2000). By means of this speciesspecific shape factor (0.06 for C. pelagicus), mean coccolith
weight was estimated from the proximal shield length (PSL),
rendering values of 159 ± 54 pg calcite for subsp. braarudii
and 120 ± 44 pg calcite for subsp. pelagicus. The main reason for this difference in calcite content estimates is that
Coccolithus coccoliths are not entirely birefringent (Beaufort, 2005; Cubillos et al., 2012), therefore calculations based
on birefringence will underestimate absolute weight but are
useful for comparative purposes.
Malformation of coccoliths was generally high in all cultures (ca. 40 % in high-P cultures at “normal” temperature)
and incomplete coccoliths also occurred frequently, especially in subsp. braarudii (ca. 30 % in high-P cultures). The
presence of malformations in control cultures can probably
be ascribed to culture artefacts which are not yet completely
understood (Langer and Benner, 2009; Langer et al., 2013b).
These authors described a high degree of malformation in
control cultures of E. huxleyi while Langer et al. (2012) reported up to 53 % malformed coccoliths in control cultures
of C. leptoporus. Although Rickaby et al. (2010) observed
low levels of background malformations (ca. 20 %) in the
same strain of subsp. braarudii as was used in this study
(RCC1200), Langer et al. (2006) described only 50 % of
coccoliths as normal in this strain, similar to our findings.
Malformations in control cultures may arise from high cell
Biogeosciences, 11, 3531–3545, 2014
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A. C. Gerecht et al.: High temperature decreases the PIC / POC ratio
densities (Langer et al., 2013b). This may explain the low
level of malformations observed by Rickaby et al. (2010) in
highly dilute batch cultures (< 2 300 cells mL−1 ) compared
to our higher cell concentrations (ca. 13 000 cells mL−1 ).
However, the 50 % malformed coccoliths reported by Langer
et al. (2006) were observed in batch cultures at cell concentrations of ca. 6 000 cells mL−1 , indicating that the percentage of malformations may not be constant over time (Langer
et al., 2013b). Another factor leading to malformations in
culture is a lack of mixing (Langer et al., 2013b). Our cultures were agitated only once a day which may have contributed to the high degree of malformations observed in our
control cultures.
Phosphorus limitation increased the percentage of malformed coccoliths further by ca. 14 %. Nutrient limitation has
previously been suggested to increase malformations in E.
huxleyi in both mesocosm and laboratory experiments (e.g.
Båtvik et al., 1997; Paasche, 1998). However, the malformation increases described by these authors are slight and/or
poorly quantified and Langer et al. (2012) argued that a difference of less than 10 % in malformations may be within
the range of natural variation. Also, these authors failed to
observe an effect of nutrient availability on coccolith morphology in C. leptoporus and concluded that the influence of
nutrient availability on the coccolith machinery is negligible.
Our study is therefore the first to show that coccolith morphology can be significantly (> 10 %) modified by limitation
by a macronutrient, in this case phosphate. In subsp. pelagicus, elevated temperature had an even stronger effect than Plimitation on coccolith morphology. The percentage of normal coccoliths in high-P cultures of subsp. pelagicus went
down to ca. 30 % at 15 ◦ C. This temperature was probably
above the temperature tolerance of this subarctic strain and
increased malformations could have been related to a stress
response to high temperature. Similarly, Langer et al. (2010)
observed a decrease of normal coccoliths in E. huxleyi from
81 % at 10 ◦ C to only 29 % at 25 ◦ C. The highest degree of
malformation in this study was observed in subsp. pelagicus
cultures faced with both P-limitation and temperature stress
indicating that several environmental stressors can add up to
compromise coccolith morphology.
5
Conclusions
The two strains of C. pelagicus, representing the two
recognized subspecies, responded in the same manner to
P-limitation, although exponential growth rates differed.
Phosphorus-limited cells of C. pelagicus had consistently
lower P-quotas, but showed no change in the PIC / POC ratio
when grown near the subspecies’ respective isolation temperature. As C. pelagicus does not produce multiple coccolith
layers, there is likely an upper constraint to the PIC / POC ratio in this species. Temperature stress, on the other hand, had
a strong impact on the PIC / POC ratio which decreased by
Biogeosciences, 11, 3531–3545, 2014
40–60 %. This was due to a reduced production of coccoliths,
whereas the mean calcite content of individual coccoliths
was not affected by experimental conditions. However, malformations of coccoliths increased under both P-limitation
and elevated temperature, with the highest percentage of
malformed coccoliths observed under a combination of Plimitation and high temperature stress. Although increased
malformations under nutrient limitation have been previously suggested (e.g. Båtvik et al., 1997; Paasche, 1998),
the evidence has so far been poor (Langer et al., 2012).
This study is therefore the first to show that limitation by
a macronutrient can indeed impact coccolith morphology.
Elevated temperature doubled P-quotas, indicating an increased P-requirement to maintain similar growth and POC
production rates at higher temperature. Thus, warmer ocean
temperatures may reinforce P-limitation in natural populations, potentially leading to lower cell densities as speciesand even strain-specific sensitivities to climate change will
contribute to changes in the abundance and composition of
phytoplankton communities (Thomas et al., 2012; Barnard
et al., 2004). This in turn will affect global biogeochemical cycles. Species- and strain-specific differences in physiology need to be taken into consideration when predicting
general responses of coccolithophores to changing environmental parameters and possible changes in CO2 feedback to
the atmosphere. It is unlikely that a more nutrient-poor ocean
will increase CO2 release by C. pelagicus as this species does
not increase calcification over POC production when faced
by nutrient limitation. However, a decrease in PIC production at elevated temperature and changes in the abundance of
this species may influence feedback mechanisms of ocean–
atmosphere CO2 exchange.
Acknowledgements. This research was funded by the Research
Council of Norway (FRIMEDBIO project 197823) and the Royal
Swedish Academy of Sciences through a grant from the Knut
and Alice Wallenberg Foundation (KAW 2009.0287). Preliminary
experiments at the Biological Station Roscoff were supported
by an FP7 ASSEMBLE research grant. We thank Sissel Brubak,
Vladyslava Hostyeva, and Viljar Skylstad for laboratory support
and Berit Kaasa for running CN analyses. We would furthermore
like to thank J.-P. Gattuso and an anonymous reviewer for their
helpful comments on the paper. A special thank you goes to Gerald
Langer, whose constructive comments greatly improved this paper.
Edited by: K. Suzuki
www.biogeosciences.net/11/3531/2014/
A. C. Gerecht et al.: High temperature decreases the PIC / POC ratio
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